Activated antigen-presenting cells (APCs) are required in order to induce a strong and long-lasting immune response. For vaccines, APCs are usually activated by either chemical or microbial components in the vaccine formulation. In contrast, normal immune responses rely upon endogenous activators of APCs, mainly CD40 ligand (CD40L, also called CD154 or TNFSF5). While numerous studies have shown how important CD40L is for vaccine responses, an ideal vaccine formulation of this protein has not been determined. Expression plasmids for CD40L result in a very strong immune response, but a soluble trimeric form of the protein is less active. This R03 Small Research Grant will test the feasibility of delivering CD40L in three different forms: as membrane CD40L, as a soluble CD40L trimer, and as a novel 12-chain, four-armed structure formed as a fusion protein with the body of surfactant protein D. Additionally, other members of the TNF superfamily of ligands have been proposed to augment immune responses but have not been tested in a HIV vaccine setting. Two of these TNFSF ligands will also be studied: RANKL (TRANCE, TNFSF11) and CD27L (CD70, TNFSF7). Each TNFSF ligand will be co-administered to mice along with a plasmid DNA construct for either HIV gp120 Env or p24 gag. Vaccine responses will be measured using assays for antibody formation, lymphoproliferative responses, cytokine production, and cytotoxity. Additionally, CD40L constructs will be combined with a fusion protein made from mycobacterial hsp70 and HIV p24 Gag to determine if CD40L has an additive effect when combined with an existing vaccine. Upon the completion of these feasibility studies, it will be clear if these concepts should be advanced into more extensive vaccine studies both for HIV and other pathogens.